Journal: bioRxiv
Article Title: Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells
doi: 10.1101/2025.08.26.672476
Figure Lengend Snippet: Downstream Processing (DSP) Scalability; Scaled DSP for MSC derived EVs and orthogonal analytical assessment of particle recovery and impurity removal. (a) Schematic representation of the scaled DSP workflow comprising clarification, tangential flow filtration (TFF), ion exchange (IEX) chromatography, post-IEX buffer exchange via TFF, and sterile filtration. Particle recoveries for each unit operation were determined by (b) -NTA, (c) Fl-NTA, and (d) Fl-FC. Protein removal throughout the entire DSP was quantified using (e) BCA assay, while impurity reduction was evaluated by (f) analytical chromatography through analysis of impurity-associated peak area under the curve (AUC). DNA removal was assessed via (g) PicoGreen™ assay. Representative chromatograms of analytical chromatography for DSP steps are shown in (j) , with EV-size peaks (9–14 min) highlighted in red, impurity peaks (14–20 min) in green, and late-eluting impurities (>20 min) in yellow. Final MSC-EVs preparation characterization included (h) scattering and (i) fluorescence NTA size distribution profiles, (k) transmission electron microscopy (TEM) imaging showing characteristic EV cup-shaped morphology, scale bar: 1 µm, and (l) Simple Western confirming EV marker expression (CD81, CD73, Syntenin) and absence of negative markers (calnexin, albumin). (m–o) Fibroblast scratch migration assay demonstrating retained bioactivity of purified MSC-EVs, quantified by cell density recovery within the wound area over 48 hour, supported by qualitative representative images, scale bar: Unless otherwise specified, Multicomparison statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001; ns not significant).
Article Snippet: The following primary antibodies were diluted to the final concentration of 0.1 μg/μL (1:10) in antibody diluent #2 (ProteinSimple, cat#042-203) and used to probe for the proteins of interest: α-CD9 (Cell Signaling, cat#50–173-3388), α-CD81 (Proteintech, cat#MAB46152,), α-Flotillin-1 (SCBT, cat#sc-74566), α-Syntenin-1 (Abcam, cat#ab133267), α-Albumin (R&D systems, cat#MAB1455), α-CD73 (R&D systems, cat#MAB57951), α-CD105 (R&D systems, cat#AF1097) and α-GAPDH (R&D systems, cat#MAB1455).
Techniques: Derivative Assay, Clarification Assay, Filtration, Chromatography, Buffer Exchange, Sterility, BIA-KA, Picogreen Assay, Fluorescence, Transmission Assay, Electron Microscopy, Imaging, Simple Western, Marker, Expressing, Migration, Purification, Comparison
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